Transporters play a central role in synaptic transmission. They are responsible for removal of neurotransmitters from the synaptic cleft and their storage in synaptic vesicles. In this project we propose to obtain mechanistic information at the molecular level on two classes of transporters. These are GLT-1 and EAAC1, the (Na+ + K+)-coupled plasma membrane transporters of the neurotransmitter glutamate, and rVMAT2, a vesicular H+-coupled monoamine transporter. Crystal structures relevant to this project have become available: GltPh and MFS transporters, bacterial homologues of GLT-1 and VMAT, respectively. In turn, now the structures provide important clues to continue our studies towards the understanding of the mechanism of transport. In this process, biochemical information is essential to validate the existing structures and those to come and to understand them in the context of function. A synergistic interaction with computational biologists also will lead to the generation of new models to be tested with our experimental tools. Using biochemical and biophysical analysis of mutants, generated by rational design or by directed evolution, we will progress towards understanding mechanism by using the available structural and biochemical information to (i) further explore the residues in the binding pocket and to modify specificities and affinities for various substrates; (ii) study the molecular determinants of ion binding and the nature of the coupling of ion and substrate fluxes and (iii) to explore the conformational transitions that occur upon ion and substrate binding. In addition to impacting on the central question of the structural basis of ion-coupled transporter function, our studies may provide important clues for the role of these transporters not only under normal physiological conditions, but also in disease. PUBLIC HEALTH RELEVANCE: Neurotransporters located in the plasma membrane and in synaptic vesicles are essential for communication between nerve cells in a process mediated by neurotransmitters. Evidence for their central role is that they are the targets of many psychoactive drugs, such as Prozac, Cocaine and Amphetamines, and that their malfunctioning leads to severe pathological conditions. Hence the study of the mode of action of the neurotransporters, as proposed in this research project, is fundamental for understanding the communication between nerve cells in the normal and diseased brain.